Abstract
It has been known for more than 50 years that the complement system is activated in autoimmune glomerulonephritis. In recent years, evidence has emerged demonstrating that uncontrolled complement activation is the primary cause of two glomerular diseases: atypical hemolytic uremic syndrome and C3 glomerulopathy. Individuals with these diseases frequently have mutations or autoantibodies that impair their ability to control complement activation. However, it is not clear why individuals with systemic defects in complement regulation often have disease that is limited to the kidneys. Therapeutic complement inhibitors are effective for treating atypical hemolytic uremic syndrome, and complement inhibitory drugs are also being developed for the treatment of other kidney diseases.
Keywords
complement, glomerulus, inflammation, immune complex, factor H, atypical hemolytic uremic syndrome, C3 glomerulopathy, eculizumab
The complement system is a group of proteins that provide an important part of the immune defense against infection. Many components of the complement system circulate as inactive proteins in the plasma. Activation of the complement system generates peptide fragments that serve as ligands for several receptors and completes a multimeric complex (C5b-9) that forms pores in membranes resulting in cell lysis.
As with all components of the immune system, proper function of the complement system helps with the effective elimination of invasive pathogens while causing minimal inflammation or injury to host tissues. However, uncontrolled activation of the complement system can cause tissue injury, and there is clear evidence that the complement cascade is activated in many autoimmune and inflammatory diseases. The kidney is particularly susceptible to complement-mediated injury, and the complement system has been implicated in the pathogenesis of multiple kidney diseases. It is also evident that acquired and congenital defects in the complement system are important risk factors for several diseases. For the most part, these disease-associated defects impair the body’s ability to regulate the complement system, thereby permitting overactivation or “dysregulation” of the complement cascade.
Uncontrolled complement alternative pathway activation appears to be central to the development of two kidney diseases: atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). These diseases are clinically and histologically distinct yet share similar risk factors. Our understanding of the pathogenesis of these diseases has been significantly advanced by recent discoveries. Although aHUS and C3G are both rare diseases, greater understanding of these diseases as extreme examples of complement dysregulation will likely provide greater insight into more common forms of glomerulonephritis that also involve complement activation.
Brief Overview of the Complement System
The complement system is composed of soluble proteins, soluble regulatory proteins, cell surface regulatory proteins, and cell surface receptors. Activation proceeds through three different pathways: the classical pathway, the mannose binding lectin (MBL) pathway, and the alternative pathway ( Fig. 21.1 ). Each pathway is activated by multiple different molecules. IgM and IgG containing immune complexes (ICs) activate the classical pathway, for example, but C-reactive protein, beta-amyloid fibrils, and other molecules also activate this pathway. The MBL pathway is activated when several different types of proteins, including MBLs, collectins, and ficolins, bind to targets on the surface of bacteria or damaged cells. All three pathways result in the cleavage of C3, forming C3b that can be covalently fixed to tissue surfaces.
Full activation of the complement cascade generates C3a, C3b, C5a, and C5b-9. Receptors for C3a and C5a induce several different inflammatory responses. Leukocytes also express receptors for C3b and the C3b inactivation fragments (iC3b and C3d). C5b-9 is also called the membrane attack complex (MAC) or the terminal complement complex (TCC). The insertion of C5b-9 in cell membranes can result in cell activation or lysis.
Several features of the alternative pathway are notable and may explain the link between activation of this pathway and kidney disease. Like the classical and MBL pathways, some proteins, including IgA, directly activate the alternative pathway. Tissue-bound C3b can combine with a protein called factor B to form an alternative pathway activating enzyme (C3bBb). Consequently, the deposition of C3b on tissue surfaces by the classical and MBL pathway can secondarily engage the alternative pathway, resulting in increased complement activation through an alternative pathway “amplification loop.” Finally, C3 in plasma is hydrolyzed at a slow rate, forming an enzyme complex that cleaves more C3 and activates the alternative pathway unless adequately controlled. This spontaneous “tickover” process continuously generates C3b that can also bind to surfaces and be amplified through the alternative pathway.
Complement Regulatory Proteins
Because the alternative pathway of complement is continuously active in plasma and tends to self-amplify, it is crucial that the body adequately controls this process. Complement activation is controlled by a group of regulatory proteins expressed on the surface of cells or that circulate in plasma. The ability of a surface to regulate alternative pathway activation determines whether the process continues to self-amplify or is terminated ( Fig. 21.2 ). These regulatory proteins provide a shield that protects the host from complement-mediated injury. Pathogens do not express complement regulatory proteins, and expression of these proteins is decreased on damaged host cells.
Specific activating proteins can trigger complement activation on a particular cell or surface, but the degree of activation is also determined by the local expression of complement regulatory proteins. Impaired regulation may lower the threshold for activation within a particular tissue, and local impairments of regulation may even be sufficient to permit spontaneous activation. Endothelial cells and podocytes each express several of the complement regulatory proteins, and ordinarily there is little evident complement activation within the glomerular capillary wall.
Several different proteins can regulate the complement system. Factor I is a circulating protein that cleaves (inactivates) C3b, forming iC3b (see Fig. 21.2 ). To function, however, factor I requires a “cofactor” protein. Several proteins with cofactor function are expressed on cell surfaces (e.g., membrane cofactor protein [MCP] and complement receptor-1 [CR1]). Factor H, a soluble alternative pathway inhibitor, also has cofactor activity. Other proteins regulate complement activation by reducing the half-life of the activating enzymes through a process termed decay acceleration. Decay accelerating factor (DAF, or CD55) is a protein linked to the surface of cells that limits complement activation, and factor H also controls complement activation by this mechanism. Several additional proteins also control the complement system through other mechanisms. For example, carboxypeptidase N is a plasma enzyme that rapidly inactivates C3a and C5a, and CD59 is a protein that is linked to cell membranes to prevent the addition of C9 during formation of C5b-9.
Congenital or acquired defects can impair the body’s ability to regulate the complement system, and these defects can increase a patient’s risk of developing different autoimmune and inflammatory diseases. Mutations have been identified in the genes for the regulatory proteins factor I, factor H, and MCP. Gain-of-function mutations have also been identified in the genes for C3 and factor B. These mutations appear to reduce the ability of the regulatory proteins to inactivate the activating enzymes, so they are functionally similar to the loss-of-function mutations seen in the complement regulatory proteins. Autoantibodies to complement proteins have also been detected in patients with various diseases, and these autoantibodies tend to impair regulation of the alternative pathway.
Defects in factor H function are the most common complement abnormalities seen in aHUS, and impaired factor H function is also seen in patients with C3G. Factor H is a soluble protein that is primarily produced in the liver and circulates in plasma at a concentration of 300 to 500 µg/mL. Factor H is made of up 20 repeating structures called short consensus repeats, or SCRs. Regulation of the alternative pathway is performed at the amino terminus of the protein in the first four SCRs, whereas the last two SCRs (19 and 20) mediate binding of factor H to molecules displayed on tissue surfaces such as glycosaminoglycans (GAGs) and sialic acid. Although it is not known why impairments in factor H so frequently manifest as injury within the glomerulus, one possibility is that the glomerular basement membrane (GBM) does not contain the other regulatory proteins, so it is completely dependent on factor H to control the alternative pathway.
The complement factor H–related proteins (CFHRs) are a group of five proteins that arose through reduplication of the gene for factor H and have high structural homology with factor H. The CFHRs all contain regions that are homologous to SCRs 19 and 20 of factor H, suggesting that they can bind similar molecules and surfaces. Various deletions and mutations in the CFHR genes have been identified in patients with aHUS and C3G. Experiments have provided conflicting data regarding the function of these proteins, but some studies suggest that the CFHRs competitively inhibit factor H and cause complement dysregulation.
Complement in Immune-Complex Glomerulonephritis
IgG and IgM containing ICs are activators of the classical pathway of complement, and there is strong evidence that the complement system is an important mediator of injury in diseases associated with glomerular IC deposition. The interaction between the complement system and ICs is complex. Some isotypes of IgG activate complement more efficiently than other isotypes. Also, the complement system helps solubilize ICs, mediating the downstream effects of ICs but also reducing their deposition in tissues. Finally, complement activation fragments affect the adaptive immune response, so the complement system may also influence disease upstream of antibody formation.
C3 Glomerulopathy
C3G is a rare form of proliferative glomerulonephritis comprising two forms: C3 glomerulonephritis (C3GN) and dense-deposit disease (DDD; previously known as type II membranoproliferative glomerulonephritis [MPGN] or MPGN type II). The unifying feature of all forms of C3G is the presence of intense C3 staining, with little or no immunoglobulin staining by immunofluorescence on kidney biopsy.
Epidemiology
Incidence of C3G is reported as one to two per million per total population. The age of onset is highly variable. The youngest reported patient was 1 year old, and about 40% of patients presented before 16 years of age in one large cohort. There was a slight prevalence of males in this series (60%), and a family history of glomerulonephritis was reported in about 11% of cases.
Etiology and Pathogenesis
A large number of molecular causes of complement alternative pathway dysregulation have been identified in patients with C3G, including autoantibodies and genetic variants that encode dysfunctional complement proteins ( Table 21.1 ).
| Complement Protein | Abnormality |
|---|---|
| C3 convertase (C3bBb) | Autoantibody
|
| Factor H | Protein
Autoantibody
Mutations
|
| Factor B | Protein
Autoantibody
Mutations
|
| Factor I | Protein
Mutations
|
| C3 | Protein
Autoantibody
Mutations
|
| CFHR1 | Mutations
|
| CHFR2 | Mutations
|
| CHFR3 | Mutations
|
| CFHR5 | Mutations
|
Autoantibodies
The most common autoantibody associated with C3G is C3 nephritic factor (C3Nef). C3Nef can be detected in approximately 70% to 80% of patients with C3G. It is more common in DDD than in C3GN and correlates with lower levels of C3. The presence of C3Nef does not correlate with clinical outcomes, however, and can be detected in healthy control subjects.
A monoclonal immunoglobulin light chain that inhibited factor H was identified in a C3G patient. This antibody blocked the regulatory function of factor H, thereby impairing regulation of the alternative pathway. Anti-factor H IgG antibodies have also been identified in additional patients with C3G. These antibodies bind to the amino terminus of the protein and block its regulatory function. Antibodies reactive to factor B and C3b have also been reported. These different autoantibodies are associated with increased alternative pathway activation, lending further support to the concept that alternative pathway activation is central to the pathogenesis of C3G. The detection of antibodies specific for the complement proteins is currently only performed in research labs and is not widely available.
Genetic Causes
Disease-associated mutations and rare variants have been identified in the genes for factor H, factor I, factor B, C3, and CFHRs 1, 2, 3, and 5 (see Table 21.1 ). An internal reduplication of a region in CFHR5 is associated with C3G, and a deletion causing formation of a hybrid CFHR2-CFHR5 protein was found in two related patients with C3G.
The different molecular causes of C3G increase alternative pathway activation or make this system resistant to regulation. It is not known, however, whether the disease is primarily caused by systemic complement activation in the plasma or local activation directly on the mesangium and glomerular capillary wall. Complement activation in the plasma could lead to deposition of complement proteins within the glomeruli as plasma is filtered through the kidney. The GBM may be dependent upon factor H for regulating the alternative pathway. Bruch’s membrane in the eye may be similarly dependent upon factor H, and patients with C3G develop retinal lesions and visual impairments.
Pathology
The identification of C3G is based on pathology and particularly on immunofluorescence ( Fig. 21.3 ). The presence of predominant C3 staining, at least twofold greater than the intensity of staining for other immune proteins (in particular IgG, IgA, IgM, C1q), is necessary for diagnosis. This definition allows for discrimination between C3G and IC-mediated forms of proliferative glomerulonephritis. Positive IF staining for C4d in equal or greater magnitude than C3 suggests activation of the classical and/or lectin pathway of complement, and it has been proposed as a metric to rule out C3 glomerulopathies.
The features of C3G by light microscopy are extremely heterogeneous. It most frequently presents with a membranoproliferative pattern, although mesangial proliferation, diffuse proliferation, and, more rarely, necrotizing lesions with extracapillary proliferation may be observed. Different patterns may coexist in the same kidney biopsy, and there may be an evolution of the lesion from mesangial proliferation to MPGN. Therefore pathologists rely on the immunofluorescence pattern to distinguish forms of C3G from other glomerular diseases that may appear similar by light microscopy. The other causes of MPGN can be idiopathic or secondary to viral infections, autoimmune diseases such as systemic lupus erythematosus, malignancies, and monoclonal gammopathies. Immunofluorescence in these forms of MPGN typically shows intense immunoglobulin staining and positive staining for C4d.
Two subtypes of C3G have been identified: DDD and C3GN. The distinction of these subtypes relies on electron microscopy (see Fig. 21.3 ). In DDD, deposits are dense, intensely osmiophilic sausage-like ribbons located within the GBM (intramembranous). The GBM becomes altered and thickened to an extent that may be visible by light microscopy. Discrete, intensely C3-positive granular deposits are also located within the mesangium. In C3GN, the C3-positive deposits are less dense, less discrete, and more diffusely located, mostly within the mesangium and on the subendothelial side of the GBM, but also in the subepithelial and intramembranous portions of the GBM. Subepithelial deposits may closely resemble the “humps” that in the past were considered pathognomonic of acute postinfectious glomerulonephritis (PIGN).
Laser microdissection of the deposits visible by electron microscopy in C3GN and DDD and proteomic analysis of their content have shown similar profiles, with no immunoglobulin but abundant components of the alternative pathway of complement. Analysis by immunofluorescence of different components of the complement pathway in kidney biopsies from patients with C3GN and DDD has also not revealed significant differences. These results confirm that these two forms of nephropathy have a shared pathogenesis and are therefore correctly classified under the common definition of C3G.
Clinical and Laboratory Features
The clinical features of C3G are extremely heterogeneous, reflecting the frequently subtle and unpredictable disease course. In one series, 41% of patients had nephrotic-range proteinuria (>3 g/day) at presentation, and 61% had microscopic hematuria. The frequency of gross hematuria was around 16% in another report. High blood pressure was present in 30.5% of patients and reduced kidney function at diagnosis in 45.5% of cases, with a mean eGFR of 69.3 mL/min/1.73 m 2 .
The clinical presentation of disease is frequently concomitant with an infectious episode. Upper respiratory tract infection was reported in 57% children with DDD. The first manifestation of disease may be gross hematuria, with recurrent episodes of gross hematuria during intercurrent infections. These patients often have persistent proteinuria and microscopic hematuria with dysmorphic red blood cells between acute episodes. This clinical picture can resemble IgA nephropathy (IgAN) and is typical but not exclusive to C3G associated with genetic alterations of CFHR5, initially described in a cluster of families from Cyprus. C3G can also resemble classic acute PIGN in which the urinary alterations appear 2 to 3 weeks after an infectious episode, frequently accompanied by hypertension and some degree of GFR loss. The associated illness is typically an upper respiratory tract infection, and the low circulating C3 with normal C4 may lead to a clinical diagnosis of PIGN. Persistently low complement levels without resolution of hematuria and proteinuria within 3 to 6 months of infection suggest a variant of C3G (sometimes called atypical postinfectious GN ).
Patients can also present with nephrotic syndrome and kidney failure. Hypertension is very frequent and may be severe, as is glomerular hematuria. The forms of C3G that present with nephrotic syndrome tend to have more intensely proliferative lesions at kidney biopsy, more severe kidney failure at onset, and poorer outcomes. More frequently, though, C3G has a subtle and remitting disease course, with no overt clinical symptoms. In such patients, microscopic hematuria and low-grade proteinuria are usually detected during routine urinalysis. In these cases, age at presentation is highly variable, and disease diagnosis may be very distant from actual disease onset. In about 10% of patients, the family history is positive for glomerulonephritis or for kidney failure of unknown origin.
Laboratory features mainly consist of low circulating C3 with normal C4 levels, reflecting activation of the alternative pathway of complement. This feature, however, is not always present and may be more frequent and intense in DDD compared with C3GN. In a report by Servais et al., low C3 plasma levels were present in 46% of all patients and 60% of those with DDD, in whom C3 levels were on average also lower. Low C4 was rare (only about 2% of cases). Therefore normal circulating C3 levels do not rule out a diagnosis of C3G. However, persistently low C3 levels with normal C4, if present, are suggestive of alternative pathway dysregulation and C3G.
A diagnosis of C3G, particularly in familial forms, warrants investigation of the alternative pathway of complement with assessment of circulating levels of different factors, measurement of C3Nef, and genetic analysis of mutations in genes coding for alternative pathway of complement proteins or regulators ( Table 21.2 ; see also Table 21.1 ).
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